FGF21 induces autophagy-mediated cholesterol efflux to inhibit atherogenesis via RACK1 up-regulation.
ABSTRACT: Fibroblast growth factor 21 (FGF21) acts as an anti-atherosclerotic agent. However, the specific mechanisms governing this regulatory activity are unclear. Autophagy is a highly conserved cell stress response which regulates atherosclerosis (AS) by reducing lipid droplet degradation in foam cells. We sought to assess whether FGF21 could inhibit AS by regulating cholesterol metabolism in foam cells via autophagy and to elucidate the underlying molecular mechanisms. In this study, ApoE-/- mice were fed a high-fat diet (HFD) with or without FGF21 and FGF21 + 3-Methyladenine (3MA) for 12 weeks. Our results showed that FGF21 inhibited AS in HFD-fed ApoE-/- mice, which was reversed by 3MA treatment. Moreover, FGF21 increased plaque RACK1 and autophagy-related protein (LC3 and beclin-1) expression in ApoE-/- mice, thus preventing AS. However, these proteins were inhibited by LV-RACK1 shRNA injection. Foam cell development is a crucial determinant of AS, and cholesterol efflux from foam cells represents an important defensive measure of AS. In this study, foam cells were treated with FGF21 for 24 hours after a pre-treatment with 3MA, ATG5 siRNA or RACK1 siRNA. Our results indicated that FGF21-induced autophagy promoted cholesterol efflux to reduce cholesterol accumulation in foam cells by up-regulating RACK1 expression. Interestingly, immunoprecipitation results showed that RACK1 was able to activate AMPK and interact with ATG5. Taken together, our results indicated that FGF21 induces autophagy to promote cholesterol efflux and reduce cholesterol accumulation in foam cells through RACK1-mediated AMPK activation and ATG5 interaction. These results provided new insights into the molecular mechanisms of FGF21 in the treatment of AS.
Project description:Macrophage-derived foam cells are a major component of atherosclerotic plaques and have an important role in the progression of atherosclerotic plaques, thus posing a great threat to human health. Photodynamic therapy (PDT) has emerged as a therapeutic strategy for atherosclerosis. Here, we investigated the effect of PDT mediated by upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) on the cholesterol efflux of THP-1 macrophage-derived foam cells and explored the possible mechanism of this effect. First, we found that PDT notably enhanced the cholesterol efflux and the induction of autophagy in both THP-1 and peritoneal macrophage-derived foam cells. The autophagy inhibitor 3-methyladenine and an ATG5 siRNA significantly attenuated PDT-induced autophagy, which subsequently suppressed the ABCA1-mediated cholesterol efflux. Furthermore, the reactive oxygen species (ROS) produced by PDT were responsible for the induction of autophagy, which could be blocked by the ROS inhibitor N-acetyl cysteine (NAC). NAC also reversed the PDT-induced suppression of p-mTOR and p-Akt. Therefore, our findings demonstrate that PDT promotes cholesterol efflux by inducing autophagy, and the autophagy was mediated in part through the ROS/PI3K/Akt/mTOR signaling pathway in THP-1 and peritoneal macrophage-derived foam cells.
Project description:Impaired autophagy in macrophages accompanies the progression of atherosclerosis and contributes to lipid loading in plaques and ineffective lipid degradation. Therefore, evoking autophagy and its associated cholesterol efflux may provide a therapeutic treatment for atherosclerosis. In the present study, berberine-mediated sonodynamic therapy (BBR-SDT) was used to induce autophagy and cholesterol efflux in THP-1 macrophages and derived foam cells. Following BBR-SDT, autophagy was increased in the macrophages, autophagy resistance in the foam cells was prevented, and cholesterol efflux was induced. The first two effects were blocked by the reactive oxygen species scavenger, N-acetyl cysteine. BBR-SDT also reduced the phosphorylation of Akt and mTOR, two key molecules in the PI3K/AKT/mTOR signaling pathway, which is responsible for inducing autophagy. Correspondingly, treatment with the autophagy inhibitor, 3-methyladenine, or the PI3K inhibitor, LY294002, abolished the autophagy-induced effects of BBR-SDT. Furthermore, induction of cholesterol efflux by BBR-SDT was reversed by an inhibition of autophagy by 3-methyladenine or by a small interfering RNA targeting Atg5. Taken together, these results demonstrate that BBR-SDT effectively promotes cholesterol efflux by increasing reactive oxygen species generation, and this subsequently induces autophagy via the PI3K/AKT/mTOR signaling pathway in both 'normal' macrophages and lipid-loaded macrophages (foam cells). Thus, BBR-SDT may be a promising atheroprotective therapy to inhibit the progression of atherosclerosis and should be further studied.
Project description:<h4>Aim</h4>Betulin is a pentacyclic triterpenoid isolated from the bark of yellow and white birch trees with anti-cancer and anti-malaria activities. In this study we examined the effects of betulin on atherosclerosis in apoE<sup>-/-</sup> mice and the underlying mechanisms.<h4>Methods</h4>Murine macrophage RAW264.7 cells and human monocyte-derived THP-1 cells were tested. Foam cell formation was detected with Oil Red O staining. Cholesterol efflux was assessed using [<sup>3</sup>H]-cholesterol efflux assay. The expression of ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) was examined using RT-PCR and Western-blotting. The ABCA1 promoter activity was evaluated using luciferase activity assay. Male apoE<sup>-/-</sup> mice fed on a high-fat-diet (HFD), and received betulin (20 and 40 mg·kg<sup>-1</sup>·d<sup>-1</sup>, ig) for 12 weeks. The macrophage content and ABCA1 expression in the aortic sinuses were evaluated with immunofluorescence staining. The hepatic, intestinal and fecal cholesterol were also analyzed in the mice.<h4>Results</h4>In RAW264.7 cells, betulin (0.1-2.5 ?g/mL) dose-dependently ameliorated oxLDL-induced cholesterol accumulation and enhanced cholesterol efflux. In both RAW264.7 and THP-1 cells, betulin increased the expression of ABCA1 and ABCG1 via suppressing the transcriptional repressors sterol-responsive element-binding proteins (SREBPs) that bound to E-box motifs in ABCA1 promoter, whereas E-box binding site mutation markedly attenuated betulin-induced ABCA1 promoter activity. In HFD-fed apoE<sup>-/-</sup> mice, betulin administration significantly reduced lesions in en face aortas and aortic sinuses. Furthermore, betulin administration significantly increased ABCA1 expression and suppressed macrophage positive areas in the aortic sinuses. Moreover, betulin administration improved plasma lipid profiles and enhanced fecal cholesterol excretion in the mice.<h4>Conclusion</h4>Betulin attenuates atherosclerosis in apoE<sup>-/-</sup> mice by promoting cholesterol efflux in macrophages.
Project description:Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.
Project description:Autophagy in macrophages plays a key role in the pathogenesis and progression of atherosclerosis and has become a potential therapeutic target. Here we show that cordycepin (Cpn), a natural derivative of adenosine, markedly reduced atherosclerotic plaque and ameliorated associated symptoms such as dyslipidemia, hyperglycemia and inflammation in ApoE-/- mice. Supplementation of Cpn dose-dependently inhibited oxLDL-elicited foam cell formation and modulated intracellular cholesterol homeostasis by inhibiting cholesterol uptake and promoting cholesterol efflux in RAW264.7 macrophages. Notably, Cpn exhibited significant stimulating effect on macrophage autophagy, as estimated by western blotting, immunofluorescent staining and autophagic vacuoles observation by transmission electron microscopy. The inhibitive effects of Cpn on foam cell formation were dramatically deteriorated in the presence of various autophagy inhibitors, suggesting that autophagy participate, at least in part, in the atheroprotective role of Cpn. Further investigations using different autophagy inhibitors and specific siRNAs for AMP-activated protein kinase (AMPK) gamma1 subunit indicated that Cpn may stimulate macrophage autophagy through AMPK-mTOR pathway. Together, our results demonstrated Cpn as a potential therapeutic agent for the prevention and treatment of atherosclerosis, and the autophagic activity presents a novel mechanism for Cpn-mediated atheroprotection.
Project description:In advanced atherosclerotic plaques, defective efferocytosis of apoptotic foam cells and decreased cholesterol efflux contribute to lesion progression. In our previous study, we demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (SDT) could induce foam cells apoptosis via the mitochondrial-caspase pathway. In the current research, we sought to explore ALA-SDT-induced apoptosis of phagocytes and the effects of cholesterol efflux and efferocytosis in advanced apoE-/- mice plaque. Methods: apoE-/- mice fed western diet were treated with ALA-SDT and sacrificed at day 1, day 3, day 7 and day 28 post treatment. THP-1 macrophage-derived foam cells were treated with ALA-SDT. 5 hours later, the supernatant was collected and added to fresh foam cells (phagocytes). Then, the lipid area, efferocytosis, cholesterol efflux, anti-inflammatory reactions and PPAR?-LXR?-ABCA1/ABCG1 pathway were detected in plaque in vivo and in phagocytes in vitro. Results: We found that ALA-SDT induced foam cells apoptosis coupled with efferocytosis and upregulation of Mer tyrosine kinase (MerTK) both in vivo and in vitro. The lipid content in plaque decreased as early as 1 day after ALA-SDT and this tendency persisted until 28 days. The enhancement of phagocytes cholesterol efflux was accompanied by an approximately 40% decrease in free cholesterol and a 24% decrease in total cholesterol in vitro. More importantly, anti-inflammatory factors such as TGF? and IL-10 were upregulated by ALA-SDT treatment. Finally, we found that PPAR?-LXR?-ABCA1/ABCG1 pathway was activated both in vivo and in vitro by ALA-SDT, which could be blocked by PPAR? siRNA. Conclusions: Activation of PPAR?-LXR?-ABCA1/ABCG1 pathway induced by ALA-SDT treatment engages a virtuous cycle that enhances efferocytosis, cholesterol efflux and anti-inflammatory reactions in advanced plaque in vivo and in phagocytes in vitro.
Project description:Genetic deficiency or inhibition of cholesteryl ester transfer protein (CETP) leads to a marked increase in plasma levels of large HDL-2 particles. However, there is concern that such particles may be dysfunctional in terms of their ability to promote cholesterol efflux from macrophages. Recently, the ATP-binding cassette transporter ABCG1, a macrophage liver X receptor (LXR) target, has been shown to stimulate cholesterol efflux to HDL. We have assessed the ability of HDL from subjects with homozygous deficiency of CETP (CETP-D) to promote cholesterol efflux from macrophages and have evaluated the role of ABCG1 and other factors in this process. CETP-D HDL-2 caused a 2- to 3-fold stimulation of net cholesterol efflux compared with control HDL-2 in LXR-activated macrophages, due primarily to an increase in lecithin:cholesterol acyltransferase-mediated (LCAT-mediated) cholesteryl ester formation in media. Genetic knockdown or overexpression of ABCG1 showed that increased cholesterol efflux to CETP-D HDL was ABCG1 dependent. LCAT and apoE contents of CETP-D HDL-2 were markedly increased compared with control HDL-2, and increased cholesterol esterification activity resided within the apoE-HDL fraction. Thus, CETP-D HDL has enhanced ability to promote cholesterol efflux from foam cells in an ABCG1-dependent pathway due to an increased content of LCAT and apoE.
Project description:Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE(-/-) mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE(-/-) mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1?, IL-6, and TNF-? levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE(-/-) mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE(-/-) mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent.
Project description:Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume.Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet.APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls.In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume.
Project description:RACK1, which was first demonstrated as a substrate of PKC? II, functions as a scaffold protein and associates with the 40S small ribosomal subunit. According to previous reports, ribosomal RACK1 was also suggested to control translation depending on the status in translating ribosome. We here show that RACK1 knockdown induces autophagy independent of upstream canonical factors such as Beclin1, Atg7 and Atg5/12 conjugates. We further report that RACK1 knockdown induces the association of mRNAs of LC3 and Bcl-xL with polysomes, indicating increased translation of these proteins. Therefore, we propose that the RACK1 depletion-induced autophagy is distinct from canonical autophagy. Finally, we confirm that cells expressing mutant RACK1 (RACK1R36D/K38E) defective in ribosome binding showed the same result as RACK1-knockdown cells. Altogether, our data clearly show that the depletion of ribosomal RACK1 alters the capacity of the ribosome to translate specific mRNAs, resulting in selective translation of mRNAs of genes for non-canonical autophagy induction.